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Open Access Highly Accessed Methodology article

A plasmid-based system for expressing small interfering RNA libraries in mammalian cells

Ajamete Kaykas and Randall T Moon*

Author affiliations

Howard Hughes Medical Institute, Department of Pharmacology, and Center for Developmental Biology, University of Washington School of Medicine, Seattle, WA 98195, USA

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Citation and License

BMC Cell Biology 2004, 5:16  doi:10.1186/1471-2121-5-16

Published: 30 April 2004

Abstract

Background

RNA interference (RNAi) is an evolutionarily conserved process that functions to inhibit gene expression. The use of RNAi in mammals as a tool to study gene function has rapidly developed in the last couple of years since the discovery that the function-inhibiting units of RNAi are short 21–25 nt double-stranded RNAs (siRNAs) derived from their longer template. The use of siRNAs allows for gene-specific knock-down without induction of the non-specific interferon response in mammalian cells. Multiple systems have been developed to introduce siRNAs into mammals. One of the most appealing of these techniques is the use of vectors containing polymerase III promoters to drive expression of hairpin siRNAs. However, there are multiple limitations to using hairpin siRNA vectors including the observation that some are unstable in bacteria and are difficult to sequence.

Results

To circumvent the limitation of hairpin siRNA vectors we have developed a convergent opposing siRNA expression system called pHippy. We have generated pHippy vectors or expression cassettes that knock down the expression of both reporter and endogenous genes. As a proof of principle that pHippy can be used to generate random siRNA libraries, we generated a small siRNA library against PGL3 luciferase and demonstrated that we could recover functional siRNAs that knock down PGL3 luciferase.

Conclusions

siRNA is a powerful tool to study gene function. We have developed a new vector with opposing convergent promoters for the expression of siRNAs, which can be used to knock down endogenous genes in a high throughput manner or to perform functional screening with random or cDNA-derived siRNA libraries.